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Prokaryotic genes related to the oxygenase domain of mammalian nitric oxide synthases (NOSs) have recently been identified. Although they catalyze the same reaction as the eukaryotic NOS oxygenase domain, their biological function(s) are unknown. In order to explore rationally the biochemistry and evolution of the prokaryotic NOS family, we have determined the crystal structure of SANOS, from methicillin-resistant Staphylococcus aureus (MRSA), to 2.4 A. Haem and S-ethylisothiourea (SEITU) are bound at the SANOS active site, while the intersubunit site, occupied by the redox cofactor tetrahydrobiopterin (H(4)B) in mammalian NOSs, has NAD(+) bound in SANOS. In common with all bacterial NOSs, SANOS lacks the N-terminal extension responsible for stable dimerization in mammalian isoforms, but has alternative interactions to promote dimer formation.
The secondary structure of bacterial RNase P RNA, a ribozyme responsible for the maturation of the 5' end of tRNAs, is well established on the basis of sequence comparison analysis. RNase P RNA secondary structures fall into two types, A and B, which share a common core formed by the assembly of two main folding domains, but differ in their peripheral elements.A revised alignment of 137 available sequences reveals new covariations allowing for the refinement of both types of secondary structures. Phylogenetic evidence is thus provided for the extension of stems P11, P14, P19, P10.1 and P15.1 through further canonical base-pairs or GAellipsisGA mismatches. These refinements led in turn to a new organization of the catalytic core, with coaxial stackings of helices P2 and P19 as well as P1 and P4. New inter-domain tertiary interactions involve loop L9 and helix P1 and loop L8 with helix P4. These features were incorporated into atomic-scale 3D models of RNase P RNA for representatives of each structural type, namely Escherichia coli and Bacillus subtilis. In each model, the juxtaposition of the core helices creates a cradle onto which the pre-tRNA substrate binds with most evolutionarily conserved residues converging towards the cleavage site. The inner cores of both types are stabilized similarly, albeit by different peripheral elements, emphasizing the modular and hierarchical organisation of the architecture of RNase P RNAs. Similarities are thus apparent between the type A modules, P16/P17/P6 and P13/P14, and their type B analogs, P5.1/P15.1 and P10. 1/P10.1a, respectively. Other noteworthy features of these models include compactness and good agreement with published crosslinking data.
The virulence-associated type III secretion system (T3SS) enables many Gram-negative bacterial pathogens to translocate proteins into the eukaryotic host cells that they infect. This unique protein transport process is mediated by the type III secretion apparatus (T3SA), a multisubunit membrane-spanning macromolecular assembly comprising >20 different proteins. Recent studies have identified biochemical and structural properties of the core T3SA, in addition to several components constituting this complex, with important implications for both the assembly process and the overall function of the T3SA.
Colicin B (55 kDa) is a cytotoxic protein that recognizes the outer membrane transporter, FepA, as a receptor and, after gaining access to the cytoplasmic membranes of sensitive Escherichia coli cells, forms a pore that depletes the electrochemical potential of the membrane and ultimately results in cell death. To begin to understand the series of dynamic conformational changes that must occur as colicin B translocates from outer membrane to cytoplasmic membrane, we report here the crystal structure of colicin B at 2.5 A resolution. The crystal belongs to the space group C2221 with unit cell dimensions a = 132.162 A, b = 138.167 A, c = 106.16 A. The overall structure of colicin B is dumbbell shaped. Unlike colicin Ia, the only other TonB-dependent colicin crystallized to date, colicin B does not have clearly structurally delineated receptor-binding and translocation domains. Instead, the unique N-terminal lobe of the dumbbell contains both domains and consists of a large (290 residues), mostly beta-stranded structure with two short alpha-helices. This is followed by a single long ( approximately 74 A) helix that connects the N-terminal domain to the C-terminal pore-forming domain, which is composed of 10 alpha-helices arranged in a bundle-type structure, similar to the pore-forming domains of other colicins. The TonB box sequence at the N-terminus folds back to interact with the N-terminal lobe of the dumbbell and leaves the flanking sequences highly disordered. Comparison of sequences among many colicins has allowed the identification of a putative receptor-binding domain.
The formation of diazodiphenoquinone pigments during bacterial oxidations of pyridinediols can be accounted for in terms of a free-radical mechanism similar to that operating for oxidations in vitro involving azasemiquinone radicals. Electron-spin-resonance spectra are of great value in pigment structure determinations.
Bacillus subtilis SpoIIE is a Ser protein phosphatase whose action on the phosphoprotein SpoIIAA triggers the cell type-specific activation of a sporulation transcription factor. Here we report that SpoIIE displays sequence similarity to the PP2C family of eukaryotic Ser/Thr protein phosphatases, and that residues common to these proteins are required for the function of both SpoIIE and TPD1, a yeast PP2C. These findings suggest that SpoIIE and the PP2C protein phosphatases are structurally related, and reveal a striking formal similarity between the SpoIIAA regulatory circuit and that of mammalian mitochondrial pyruvate dehydrogenase. This similarity may reflect an evolutionarily conserved mechanism of biological regulation based on the interplay of His protein kinase-like Ser kinases and PP2C-like protein phosphatases.
Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis, by transferring an adenylyl group from ATP to 4'-phosphopantetheine, yielding dephospho-CoA (dPCoA). Each phosphopantetheine adenylyltransferase (PPAT) subunit displays a dinucleotide-binding fold that is structurally similar to that in class I aminoacyl-tRNA synthetases. Superposition of bound adenylyl moieties from dPCoA in PPAT and ATP in aminoacyl-tRNA synthetases suggests nucleophilic attack by the 4'-phosphopantetheine on the alpha-phosphate of ATP. The proposed catalytic mechanism implicates transition state stabilization by PPAT without involving functional groups of the enzyme in a chemical sense in the reaction. The crystal structure of the enzyme from Escherichia coli in complex with dPCoA shows that binding at one site causes a vice-like movement of active site residues lining the active site surface. The mode of enzyme product formation is highly concerted, with only one trimer of the PPAT hexamer showing evidence of dPCoA binding. The homologous active site attachment of ATP and the structural distribution of predicted sequence-binding motifs in PPAT classify the enzyme as belonging to the nucleotidyltransferase superfamily.
The key role of telluric microorganisms in pesticide degradation is well recognized but the possible relationships between the biodiversity of soil microbial communities and their functions still remain poorly documented. If microorganisms influence the fate of pesticides, pesticide application may reciprocally affect soil microorganisms. The objective of our work was to estimate the impact of 2,4-D application on the genetic structure of bacterial communities and the 2,4-D-degrading genetic potential in relation to 2,4-D mineralization. Experiments combined isotope measurements with molecular analyses. The impact of 2,4-D on soil bacterial populations was followed with ribosomal intergenic spacer analysis. The 2,4-D degrading genetic potential was estimated by real-time PCR targeted on tfdA sequences coding an enzyme specifically involved in 2,4-D mineralization. The genetic structure of bacterial communities was significantly modified in response to 2,4-D application, but only during the intense phase of 2,4-D biodegradation. This effect disappeared 7 days after the treatment. The 2,4-D degrading genetic potential increased rapidly following 2,4-D application. There was a concomitant increase between the tfdA copy number and the 14C microbial biomass. The maximum of tfdA sequences corresponded to the maximum rate of 2,4-D mineralization. In this soil, 2,4-D degrading microbial communities seem preferentially to use the tfd pathway to degrade 2,4-D.
The effects of cyanogenic Pseudomonas fluorescens strains introduced into soil on the kinetic of colony formation and bacterial community structure were investigated. About 7.8 x 10(8) and 1.2 x 10(9) cfu per g dry soil of TA1 and B2 were added to the soil portions, respectively. The parameters of colony formation by heterotrophic soil bacteria were determined. The bacterial community structure and phenotypic diversity were studied using concept of r/K strategies and echophysiological index, respectively. The physiological state of indigenous heterotrophic bacteria and gram-negative group did not change under the influence of the cyanogenic strains introduced. Phenotypic diversity of the soil bacteria also did not change significantly. However, some short-term shifts in community structure of indigenous heterotrophic bacteria were noticed. This study shows that the introduction of great numbers of cyanogenic P. fluorescens strains could be safely used as potential agents in biological control of soil-born pathogens.
On mild acid degradation of a lipopolysaccharide from Pseudomonas cepacia strain IMV 4137, a serologically active O-specific polysaccharide was obtained and shown to contain L-rhamnose and D-galactose. According to 1H- and 13C-NMR data as well as methylation analysis, the polysaccharide is made up of disaccharide repeating units of the following structure:----2)-alpha-L-Rhap-(1----4)-alpha-D-Galp-(1----.
GE37468 A is a novel antibiotic produced by Streptomyces sp. ATCC 55365. It has molecular mass 1309.48 and formula C59H52O12N14S5 and belongs to the thiazolyl peptide group of antibiotics. The structure was elucidated by 1H and 13C NMR and MS studies on intact molecule and its hydrolysis products. The antibiotic is a highly modified peptide containing a macrocycle and a side chain composed of a thiazole ring and two dehydroalanine units.
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Uropathogenic Escherichia coli produce heteropolymeric surface fibers called P pili, which present an adhesin at their tip that specifically recognizes globoside receptors on the host uroepithelium. The initial attachment step is thought to be essential for pathogenesis. P pili are composite fibers consisting of a thin tip fibrillum joined end to end to a rigid helical rod. Here we show that the ordered assembly of these structures requires the activity of two proteins that are minor components of the tip fibrillum, PapF and PapK. PapF is required for the correct presentation of the adhesin at the distal end of the tip fibrillum. PapK regulates the length of the tip fibrillum and joins it to the pilus rod. We propose that these subunits function as adaptors, by providing complementary surfaces to different substructures of the pilus and promoting their proper associations. In addition, the conversion of chaperone-subunit complexes into pili depends on PapF and PapK since a papF- papK- double mutation abolishes piliation. We suggest that in addition to the adaptor functions of PapF and PapK, they are also required to initiate the formation of tip fibrillae and pilus rods.
Type IV pili are cell surface organelles found on many Gram-negative bacteria. They mediate a variety of functions, including adhesion, twitching motility, and competence for DNA uptake. The type IV pilus is a helical polymer of pilin protein subunits and is capable of rapid polymerization or depolymerization, generating large motor forces in the process. Here we show that a specific interaction between the outer membrane secretin PilQ and the type IV pilus fiber can be detected by far-Western analysis and sucrose density gradient centrifugation. Transmission electron microscopy of preparations of purified pili, to which the purified PilQ oligomer had been added, showed that PilQ was uniquely located at one end of the pilus fiber, effectively forming a "mallet-type" structure. Determination of the three-dimensional structure of the PilQ-type IV pilus complex at 26-angstroms resolution showed that the cavity within the protein complex was filled. Comparison with a previously determined structure of PilQ at 12-angstroms resolution indicated that binding of the pilus fiber induced a dissociation of the "cap" feature and lateral movement of the "arms" of the PilQ oligomer. The results demonstrate that the PilQ structure exhibits a dynamic response to the binding of its transported substrate and suggest that the secretin could play an active role in type IV pilus assembly as well as secretion.
To investigate the responses of Baltic Sea wintertime bacterial communities to changing salinity (5 to 26 practical salinity units), an experimental study was conducted. Bacterial communities of Baltic seawater and sea ice from a coastal site in southwest Finland were used in two batch culture experiments run for 17 or 18 days at 0 degrees C. Bacterial abundance, cell volume, and leucine and thymidine incorporation were measured during the experiments. The bacterial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNA genes with sequencing of DGGE bands from initial communities and communities of day 10 or 13 of the experiment. The sea ice-derived bacterial community was metabolically more active than the open-water community at the start of the experiment. Ice-derived bacterial communities were able to adapt to salinity change with smaller effects on physiology and community structure, whereas in the open-water bacterial communities, the bacterial cell volume evolution, bacterial abundance, and community structure responses indicated the presence of salinity stress. The closest relatives for all eight partial 16S rRNA gene sequences obtained were either organisms found in polar sea ice and other cold habitats or those found in summertime Baltic seawater. All sequences except one were associated with the alpha- and gamma-proteobacteria or the Cytophaga-Flavobacterium-Bacteroides group. The overall physiological and community structure responses were parallel in ice-derived and open-water bacterial assemblages, which points to a linkage between community structure and physiology. These results support previous assumptions of the role of salinity fluctuation as a major selective factor shaping the sea ice bacterial community structure.
Crystal structures of bacterial CLC proteins were solved recently, but it is unclear to which level of detail they can be extrapolated to mammalian chloride channels. Exploiting the difference in inhibition by 9-anthracene carboxylic acid (9-AC) between ClC-0, -1, and -2, we identified a serine between helices O and P as crucial for 9-AC binding. Mutagenesis based on the crystal structure identified further residues affecting inhibitor binding. They surround a partially hydrophobic pocket close to the chloride binding site that is accessible from the cytoplasm, consistent with the observed intracellular block by 9-AC. Mutations in presumably Cl--coordinating residues yield additional insights into the structure and function of ClC-1. Our work shows that the structure of bacterial CLCs can be extrapolated with fidelity to mammalian Cl- channels.